Abstract A crucial first step in DNA repair involves the recognition of genome damage, which in turn activates signaling pathways that recruit effectors and resolve the lesion. However, whether this ?sensor- transducer-mediator? paradigm is generally applicable to pathways dedicated to repairing each distinct type of DNA lesion remains unknown. Understanding the signaling events that mediate the recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment. Our preliminary studies demonstrate that human cells have a heretofore unrecognized repair signaling pathway that is highly specific for recruiting alkylation repair factors to nuclear foci and link this pathway to an inherited human disease. Since multiple alkylation repair factors appear to be recruited to these foci, we have termed them nuclear SCARs (Specialized Centers for Alkylation Repair). In this proposal, we seek to understand whether recruitment of alkylation repair factors to these sites is critical for resolution of alkylation repair (Aim 1). We will characterize the upstream E3 ubiquitin ligase, RNF113A, which we have found to play a central role in this pathway and is mutated in the progeroid syndrome trichothiodystrphy (Aim 2). Finally, we will test whether targeting this pathway could promote chemosensitization in human tumor models (Aim 3). These studies will greatly increase our understanding of how cells detect and activate DNA repair pathways. Since alkylation repair is critical for reversing the toxic effects of many chemotherapy agents, our work will provide new insights into how cell respond to such therapy, and may reveal several novel molecular targets for chemosensitization.